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Zika virus (ZIKV), a mosquito-borne flavivirus, is a global health concern because of its association with severe neurological disorders such as neonatal microcephaly and adult Guillain-Barre syndrome. Although many efforts have been made to combat ZIKV infection, there is currently no approved vaccines or antiviral drugs available and there is an urgent need to develop effective anti-ZIKV agents. In this study, 26 acetylarylamine-S-DACOs derivatives were prepared, and eight of them were found to have inhibitory activity against Zika virus. Among these substances, 2-[(4-cyclohexyl-5-ethyl-6-oxo-1,6-dihydropyrimidin-2-yl)thio]-N-(3,5-difluorophenyl)acetamide ( 4w ) with the best anti-ZIKV activity was selected for in-depth study of antiviral activity and mechanism of action. Here, we discovered 4w targeted on the ZIKV NS5 RNA -dependent RNA polymerase (RdRp), which exhibited good in vitro antiviral activity without cell species specificity, both at the protein level and at the RNA level can significantly inhibit ZIKV replication. Preliminary molecular docking studies showed that 4w preferentially binds to the palm region of NS5A RdRp through hydrogen bonding with residues such as LYS468, PHE466, GLU465, and GLY467. ZIKV NS5 RdRp enzyme activity experiment showed that 4w could directly inhibit ZIKV RdRp activity with EC 50 = 11.38 ± 0.51 μM. In antiviral activity studies, 4w was found to inhibit ZIKV RNA replication with EC 50 = 6.87 ± 1.21 μM. ZIKV-induced plaque formation was inhibited with EC 50 = 7.65 ± 0.31 μM. In conclusion, our study disclosed that acetylarylamine-S-DACOs is a new active scaffolds against ZIKV, among which compound 4w was proved to be a potent novel anti-ZIKV compound target ZIKV RdRp protein. These promising results provide a future prospective for the development of ZIKV RdRp inhibitors.
5-Alkyl-6-aryl-2-(phenylaminocarbonylmethylthio)pyrimidin-4(3H)-ones (acetylarylamine-S-DABOs general structure 1 , ) are a class of excellent non-nucleoside reverse transcriptase inhibitors (NNRTIs) with high broad-spectrum HIV-1 inhibitory activity ( Yu et al., 2009 ; Yu et al., 2011 ). In our previous antiviral studies, starting from compounds of general structure 1 , we replaced the aromatic ring at the C-6 position of the pyrimidine by a cyclohexyl moiety, obtaining a series of 5-Alkyl-6-cyclohex-yl-2-(phenylaminocarbonylmethylthio)pyrimidin-4(3H)-ones (acetylarylamine-S- DACOs general structure 2 , ) which can inhibit HIV replication in the low nanomolar range ( He et al., 2011 ), while compounds of general structure 3 showed good anti-HCV activity ( He et al., 2015 ). In recent years, we have launched our search of new active scaffolds against flaviviruses such as HCV, DENV and ZIKV ( Wu et al., 2017 ; Qian et al., 2022 ; Rui et al., 2022 ). Given the success of drug development against HCV infection and the presence of HCV homologs with ZIKV, the repurposing of HCV inhibitors for ZIKV is an attractive starting point for the discovery of a novel skeleton of anti-ZIKV drugs. By comparing the structures of the 2 and 3 series compounds, it can be seen that the compounds with F or OCH 3 substituent on the benzene ring at the end of C-2 side chain have good anti-HIV or HCV activity. Therefore, in the design of target molecules, fluoro-containing substituents such as F, CF 3 , OCF 3 and OCH 3 were selected and introduced into different positions on the benzene ring. In addition, the C-6 substituent also had a significant effect on the antiviral activity of the compounds. For example, C-6 cyclohexylmethyl substituted compounds 2 have anti-HIV activity, while C-6 cyclohexyl substituted compounds 3 have anti-HCV activity. Therefore, we intend to synthesize C-6 cyclohexyl or C-6 cyclohexylmethyl substituted acetylaryl-amine-S-DACOs target molecules simultaneously. Therefore, using compounds of general structures 2 and 3 as lead compounds, a series of new acetylarylamine-S-DACO derivatives 4a-z ( ) were designed by introducing selective substituents (R) on the ω-phenyl group of the pyrimidine ring C-2 side chain and a cyclohexyl or cyclohexyl methyl at the C-6 position. In this paper, we describe the synthesis, cell-based and target-based activity evaluation of the series of acetylarylamine-S-DACOs derivatives against ZIKV infection.
Initially, the novel acetylarylamine-S-DACOs 4a-z were tested for their cytotoxicity and anti-ZIKV activity in Vero cells by plaque formation according to previously detailed procedure (Xu et al., 2017). The activity data was interpreted in CC50 values (cytotoxicity), EC50 (anti-ZIKV activity) and SI (selectivity index, given by the CC50/EC50 ratio) ( ). As show in , the preliminary plaque results revealed that there are eight active compounds against ZIKV replication with EC50 < 20 μM and CC50 > 90 μM, ensuring that antiviral effect of these compounds is not related to their toxicity. The most potent compounds were 4j, 4n, and 4w with EC50 values of 7.48, 7.75, and 7.65 μM, respectively. They were about 6 times more potent than the reference drug Ribavirin (EC50 = 48.88 μM). From , we can make a preliminary summary of this series of compounds: First, the type of substituent R on benzene ring has a significant impact on the anti-ZIKV activity of these compounds. For example, the R of the above eight compounds showing anti-ZIKV were all fluorine substituents, while the compounds 4a-f, 4y, and 4z obtained by introducing methoxyl onto benzene ring did not show activity. In addition, the position of the substituent R on the benzene ring also has an important influence on the activity. For example, the activity of the 3′-F-substituted compound 4j (EC50 = 7.48 μM) is better than that of the 2′-F-substituted analog 4h (EC50 = 10.77 μM), while the 4′-F-substituted analog 4l becomes inactive compound. The 3′-OCF3-substituted compound 4r can inhibit ZIKV with EC50 value of 8.84 μM, while its 4′-OCF3-substituted counterpart 4p is inactive against ZIKV. At last, the length (n) of the connecting carbon chain between the C-6 site of the pyrimidine ring and cyclohexane also affects the activity of the compound, and there may be a synergistic relationship between n and substituent R. For example, compound 4j (R = 3′-F, n = 1), 4n (R = 4′-CF3, n = 1), and 4r (R = 3′-OCF3, n = 1) are more active than their counterpart 4i (R = 3′-F, n = 0), 4m (R = 4′-CF3, n = 0), and 4q (R = 3′-OCF3, n = 0), respectively, showing cyclohexylmethyl (n = 1) is more favorable for activity. Unlike SAR of 3′-F or 3′-OCF3 series, 4x (R = 3′,5′-diF, n = 1) is less active than 4w (R = 3′,5′-diF, n = 0), showing cyclohexyl (n = 0) is more favorable for activity.
The strongest plaque inhibition by compound 4w is shown in . As we can see from , 4w significantly inhibited the plaque formation of ZIKV, and this inhibitory effect was positively correlated with the concentration of 4w. It inhibited ZIKV plaque formation with EC50 = 7.65 ± 0.31 μM ( ). We tested its cytotoxicity to A549, Huh7 and Vero cells by MTT method, and found that it had no obvious toxicity to these three different cell lines, the CC50 was greater than 200 μM ( ), and its SI value was greater than 26, which suggested that 4w could be used as a new type of anti-ZIKV active compound for further development and research. Subsequently, compound 4w, as a novel anti-ZIKV skeleton, was subjected to further analysis to detect the antiviral effects and elucidate the drug target and the mode of drug action.
Open in a separate windowAmong the viral targets, NS5 polymerase is one of the most promising and exploited targets, being highly conserved among flaviviruses. Previously, some related pyrimidones have been identified as potential HCV inhibitors targeting NS5 RdRp. For example, Ding et al. have reported 5-cyano-6-aryl-2-thiouracil (compound A, ) as a potent inhibitor of HCV NS5B RdRp (IC50 = 3.8 μM) (Ding et al., 2006). Wu et al. (2017) identified 2-hydroxylphenethyl sulfanyl-oxopyrimidines derivatives as potential anti-HCV agent targeting HCV NS5B RdRp (e.g., compound B, ). After our research group revealed the potent anti-HCV activity of acetylarylamine-S-DACO derivatives (general structure 3, ), Shen (2015) further determined that this series of compounds targeted HCV NS5B RdRp. For example, compound 4x and 4w ( ) as a potent inhibitor of HCV NS5B RdRp have IC50 of 0.089 μΜ and 0.102 μM. Since compound 4w has already been shown to be a HCV RdRp inhibitor, we speculated compound 4w may also act on ZIKV RdRp.We first detected whether 4w could interact with ZIKV RdRp by molecular docking method, and found that 4w can bind to ZIKV RdRp domain with low free energy ( ). ZIKV RdRp is mainly composed of three domains, namely palm domain (amino acid regions 321–488 and 542–608), finger domain (amino acid regions 489–541 and 609–714) and thumb (amino acid regions 715–903) (Chen et al., 2021). In the docking results, it was found that 4w mainly binds to the palm domain of ZIKV RdRp, and preferentially binds to LYS468, PHE466, GLU465, and GLY467 amino acids in the palm region by hydrogen bonding force ( ). Details are as follows: 1) The pyrimidine ring of compound 4w is immobilized in the binding cavity by hydrogen bonding between its C-4 carbonyl group and LYS468 and π-π interaction with the benzene ring of PHE466. 2) The C-2β-Carbonyl of 4w formed triple hydrogen bonds with GLU465, PHE466 and GLY467, thus facilitating stable binding of the inhibitor to ZIKV RdRp;3) The C-2 terminal 3, 5-2F-phenyl group of 4w points to the region surrounded by the side chains of HIS732, GLU735, and ASP734. In addition to the π-π interaction between the benzene ring and the imidazole ring of HIS732, the two F atoms on the benzene ring also form the F-H interaction with the two polarized CH groups on GLU735 and ASP734, respectively, which further strengthens the combination of 4w and ZIKV RdRp.
Open in a separate windowOpen in a separate windowCell thermal shift assay (CETSA) experiments are often used to detect the binding of intracellular drugs to target proteins. The assay was based on the principle of ligand-induced changes in protein thermal stability. The stability of the protein is evaluated by comparing the melting curves between the control group and the experimental group, thus assessing the interaction of the compound with the target protein.
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Molecular docking experiments, suggested that 4w interacts with ZIKV RdRp domain, and ZIKV RdRp domain is the C-terminal subdomain of ZIKV NS5 protein. Therefore, in order to further verify the interaction between 4w and ZIKV NS5, ZIKV-infected cell were treated with 4w (at 50 μM concentration), then total protein was incubation for 60 min at room temperature, and it was divided into nine equal parts. The same volume of DMSO was added as a control, and the treated protein was heated in a temperature gradient and then detected ZIKV NS5 protein by WB assay ( ). It was found that 4w can bind to ZIKV NS5 protein in cells, thereby improving the thermal stability of ZIKV NS5 protein and preventing its aggregation and precipitation at high temperature. The apparent aggregation temperature (Tagg) of ZIKV NS5 protein was increased from 42.3°C to 49.3°C ( ).
Open in a separate windowThrough molecular docking experiments and cell thermal shift analysis, we supose that 4w may have a direct interaction with ZIKV RdRp. In order to verify whether the binding of 4w and NS5 protein is directly related to ZIKV RdRp, we expressed and purified ZIKV RdRp domain, and detected its activity according to the method described in the literature (Qian et al., 2022) ( ). Through RdRp activity assay results, we found that 4w inhibited ZIKV RdRp activity with EC50 = 11.38 ± 0.51 μM ( ). Heparin is a protein chelating agent. According to the literature (Sáez-Álvarez et al., 2019), it can significantly inhibited the activity of ZIKV RdRp. Heparin was selected as the positive control drug in this experiment, and the syto9-only group was used as the negative control, the same volume of DMSO as a positive control.
Open in a separate windowThrough above experiments, we demonstrated that 4w inhibits ZIKV RdRp activity in a dose-dependent manner. ZIKV RdRp was a important domain in the C-terminal of ZIKV NS5, and plays a crucial role in the replication and synthesis of RNA in the life cycle of ZIKV. In order to further verify that 4w activity is related to RdRp domain inhibition, we examined the effect of 4w on the replication cycle of ZIKV using four different drug treatments and time-sharing withdrawal experiments ( ). Through four different treatments with 4w, we found that 4w mainly acts on the post-entry stage of the virus, and it has a significant inhibitory effect on the viral load in cells after virus infection ( ). In the time of drug withdrawal experiment ( ), we found that drug withdrawal 4–6 h after dosing could significantly inhibit the release of progeny virus in the supernatant of ZIKV infected cells. From the results of drug treatment, we found that 4w may mainly act on the ZIKV RNA synthesis stage ( ), which once again suggesting that 4w binds into the ZIKV RdRp domain inhibition.
Open in a separate windowIn order to re-verify the inhibitory effect of 4w on ZIKV at the molecular level, we used ZIKV to infect cell lines of different origins (A549, Vero, Huh7 cells) and then treated with 4w in a concentration gradient. The change of viral load in the supernatant of cells after treatment was detected by qRT-PCR method. It was found that 4w could significantly inhibit the replication of viral RNA in different cell lines infected with ZIKV ( ). In Vero, A549 and Huh7 cells, 4w inhibited the replication of ZIKV RNA with EC50 values 6.871 ± 1.21, 5.18 ± 0.69, and 6.76 ± 0.52 μM, respectively. These results indicate that the inhibitory effect of 4w on ZIKV is not dependent on cell species specificity.
Open in a separate windowZIKV E protein is one of the most important structural proteins of ZIKV, which plays an important role in the correct assembly of the virus and evasion of the host’s innate immunity (Jaimipuk et al., 2022). We have verified that 4w has a significant inhibitory effect on ZIKV-infected cell lines from different sources at the molecular level, but it is unknown whether it has the same antiviral effect at the protein level. Therefore, in order to explore the inhibitory effect of 4w on ZIKV protein level, we added serially diluted 4w to ZIKV-infected Vero cells for 48 h, collected the total protein for WB experiments, and then detected the effect of 4w on ZIKV E and NS5 protein. At the same time, the inhibitory effect of 4w on ZIKV E protein was verified again by immunofluorescence experiment. Through WB experiment ( ), we found that 4w can still well inhibit the expression of ZIKV E and NS5 protein at the viral protein level, and its 12.5 μM can significantly inhibit the expression of ZIKV E and NS5 protein ( ). Immunofluorescence results showed that 4w 25 μM completely inhibited the expression of ZIKV E protein ( ).
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